Anaplasma phagocytophilum is an obligate intracellular bacterial pathogen that invades neutrophils and endothelial cells to cause the emerging and potentially fatal infection, human granulocytic anaplasmosis (HGA). A. phagocytophilum converts its host neutrophil into a Trojan Horse that facilitates pathogen replication and dissemination. It downregulates the neutrophil antimicrobial response, thereby raising susceptibility to opportunistic infections. Thus, blocking A. phagocytophilum infection of neutrophils would potentially prevent the stage of HGA associated with pathogen dissemination and increased risk of opportunistic infections. A. phagocytophilum infects microvascular endothelial cells of heart and liver. Endothelial cells are also implicated as the initial cell typ that A. phagocytophilum infects following inoculation via tick feeding. Infected endothelial cells are capable of transferring the bacterium to neutrophils. Therefore, abrogating A. phagocytophilum invasion of endothelial cells would potentially prevent infection of major organs and initial establishment of infection. The overall goal of this project is to identify and functioally characterize A. phagocytophilum outer membrane proteins that facilitate infection. We have identified outer membrane protein A (OmpA) and Asp14 (14-kDa Ap surface protein) as the first two A. phagocytophilum proteins that are critical for invasion of both myeloid cells and endothelial cells. In Aim 1, we will identify the OmpA and Asp14 domains that mediate A. phagocytophilum uptake and determine if either protein is sufficient for invasion. In Aim 2, we will identify the host cell receptors of OmpA and Asp14. In Aim 3, we will directly assess the relevance of OmpA and Asp14 to infectivity using novel transgenic A. phagocytophilum organisms that can be induced to express antisense RNA against ompA or asp14 to knock down OmpA or Asp14 expression. Achieving our goals will provide a robust understanding of A. phagocytophilum cellular invasion and identify the first two invasin-receptor pairs for any Anaplasmataceae pathogen. It will also yield a valuable tool for assessing gene function in obligate intracellular bacteria. Lastly, our work will potentially aid the design of novel intervenion strategies that target OmpA and Asp14 to prevent HGA by blocking multiple stages of infection.